Technical Abstract:
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s Disease (JD), a chronic enteritis that occurs in dairy cattle and other ruminants. A 2007 NAHMS Dairy Study demonstrated that over 68% of dairy herds are infected with JD so the risk of exposure within a herd is high. MAP is primarily shed in the feces but it is also shed into the milk and colostrum of infected cows. Because of this, there exists concern about transfer of the organism from dam to calf and the prevalence of MAP in the milk supply. The amount of MAP shed into milk is not well defined because the complexity of the milk matrix and presence of confounding microorganisms makes it difficult to recover MAP via culture methods. The objective of this study is to optimize the decontamination of whole raw milk for the isolation of viable MAP and compare recovery rates in liquid culture mediums. The efficacy of two liquid culture mediums, TREK-ESP and BD Bactec 12B were compared for recovery speed and thresholds, incidence of contamination, and reproducibility of results. Milk collected from a non-infected cow was spiked with known concentrations of MAP (10**2 to 10**8 cfu/ml). Two chemical decontaminates were investigated, hexadecylpyridinium chloride (HPC) and N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH). Variables investigated included concentration of these two chemical decontaminants, temperature of decontamination, centrifugation speed, and time of incubation. It was found that NALC-NaOH was the superior chemical for decontamination and that MAP recovery from milk was highest when decontaminated with 1.5% NaOH for a 15-minute exposure time. In comparing the two liquid culture mediums, Bactec 12B was superior in recovery with thresholds of less than 10**2 cfu/ml and superior in speed of recovery of viable MAP. TREK-ESP culture demonstrated an increased incidence in false positive and false negative results that were not observed in Bactec 12B medium. Optimized methods will be used to assess the frequency and level of MAP shed into milk during a complete lactation period of naturally infected dams.